Next generation sequencing for clinical diagnostics: Five year experience of an academic laboratory.

Clinical laboratories have adopted next generation sequencing (NGS) as a gold standard for the diagnosis of hereditary disorders because of its analytic accuracy, high throughput, and potential for cost-effectiveness. We describe the implementation of a single broad-based NGS sequencing assay to meet the genetic testing needs at the University of Minnesota. A single hybrid capture library preparation was used for each test ordered, data was informatically blinded to clinically-ordered genes, and identified variants were reviewed and classified by genetic counselors and molecular pathologists. We performed 2509 sequencing tests from August 2012 till December 2017. The diagnostic yield has remained steady at 25%, but the number of variants of uncertain significance (VUS) included in a patient report decreased over time with 50% of the patient reports including at least one VUS in 2012 and only 22% of the patient reports reporting a VUS in 2017 (p = .002). Among the various clinical specialties, the diagnostic yield was highest in dermatology (60% diagnostic yield) and ophthalmology (42% diagnostic yield) while the diagnostic yield was lowest in gastrointestinal diseases and pulmonary diseases (10% detection yield in both specialties). Deletion/duplication analysis was also implemented in a subset of panels ordered, with 9% of samples having a diagnostic finding using the deletion/duplication analysis. We have demonstrated the feasibility of this broad-based NGS platform to meet the needs of our academic institution by aggregating a sufficient sample volume from many individually rare tests and providing a flexible ordering for custom, patient-specific panels.

Optimization of a microfluidics-based next generation sequencing assay for clinical oncology diagnostics.

BACKGROUND: Massively parallel, or next-generation, sequencing is a powerful technique for the assessment of somatic genomic alterations in cancer samples. Numerous gene targets can be interrogated simultaneously with a high degree of sensitivity. The clinical standard of care for many advanced solid and hematologic malignancies currently requires mutation analysis of several genes in the front-line setting, making focused next generation sequencing (NGS) assays an effective tool for clinical molecular diagnostic laboratories. METHODS: We have utilized an integrated microfluidics circuit (IFC) technology for multiplex PCR-based library preparation coupled with a bioinformatic method designed to enhance indel detection. A parallel low input PCR-based library preparation method was developed for challenging specimens with low DNA yield. Computational data filters were written to optimize analytic sensitivity and specificity for clinically relevant variants. RESULTS: Minimum sequencing coverage and precision of variant calls were the two primary criteria used to establish minimum DNA mass input onto the IFC. Wet-bench and bioinformatics protocols were modified based on data from the optimization and familiarization process to improve assay performance. The NGS platform was then clinically validated for single nucleotide and indel (up to 93 base pair) variant detection with overall analytic accuracy of 98% (97% sensitivity; 100% specificity) using as little as 3 ng of formalin-fixed, paraffin-embedded DNA or 0.3 ng of unfixed DNA. CONCLUSIONS: We created a targeted clinical NGS assay for common solid and hematologic cancers with high sensitivity, high specificity, and the flexibility to test very limited tissue samples often encountered in routine clinical practice.

An innovative medical civil-military operation training program.

The San Antonio Military Pediatric Center has developed an innovative humanitarian civic assistance (HCA) program. Many medical HCA programs focus on short-term medical interventions and provide transient benefit. To have a more lasting impact, this program focuses on public health surveillance. U.S. military medics conduct random household nutritional surveys and train in austere settings and on rounds in Honduran hospitals. Since 2001, >200 military medics have been trained in population assessment, primary medical care in developing nations, and other skills critical for medical civil-military operations. All activities are coordinated with the host nation. Public health data are collected and reported to Honduran public health leaders, the U.S. Agency for International Development, and nongovernmental organizations, to assist with program and policy development. This innovative project is a potential model to improve both military training and host nation benefit from HCA programs.

Clinical validation of targeted next-generation sequencing for inherited disorders.

CONTEXT: Although next-generation sequencing (NGS) can revolutionize molecular diagnostics, several hurdles remain in the implementation of this technology in clinical laboratories. OBJECTIVES: To validate and implement an NGS panel for genetic diagnosis of more than 100 inherited diseases, such as neurologic conditions, congenital hearing loss and eye disorders, developmental disorders, nonmalignant diseases treated by hematopoietic cell transplantation, familial cancers, connective tissue disorders, metabolic disorders, disorders of sexual development, and cardiac disorders. The diagnostic gene panels ranged from 1 to 54 genes with most of panels containing 10 genes or fewer. DESIGN: We used a liquid hybridization-based, target-enrichment strategy to enrich 10 067 exons in 568 genes, followed by NGS with a HiSeq 2000 sequencing system (Illumina, San Diego, California). RESULTS: We successfully sequenced 97.6% (9825 of 10 067) of the targeted exons to obtain a minimum coverage of 20× at all bases. We demonstrated 100% concordance in detecting 19 pathogenic single-nucleotide variations and 11 pathogenic insertion-deletion mutations ranging in size from 1 to 18 base pairs across 18 samples that were previously characterized by Sanger sequencing. Using 4 pairs of blinded, duplicate samples, we demonstrated a high degree of concordance (>99%) among the blinded, duplicate pairs. CONCLUSIONS: We have successfully demonstrated the feasibility of using the NGS platform to multiplex genetic tests for several rare diseases and the use of cloud computing for bioinformatics analysis as a relatively low-cost solution for implementing NGS in clinical laboratories.

Position of the academy of nutrition and dietetics: nutrition security in developing nations: sustainable food, water, and health.

It is the position of the Academy of Nutrition and Dietetics that all people should have consistent access to an appropriately nutritious diet of food and water, coupled with a sanitary environment, adequate health services, and care that ensure a healthy and active life for all household members. The Academy supports policies, systems, programs, and practices that work with developing nations to achieve nutrition security and self-sufficiency while being environmentally and economically sustainable. For nations to achieve nutrition security, all people must have access to a variety of nutritious foods and potable drinking water; knowledge, resources, and skills for healthy living; prevention, treatment, and care for diseases affecting nutrition status; and safety-net systems during crisis situations, such as natural disasters or deleterious social and political systems. More than 2 billion people are micronutrient deficient; 1.5 billion people are overweight or obese; 870 million people have inadequate food energy intake; and 783 million people lack potable drinking water. Adequate nutrient intake is a concern, independent of weight status. Although this article focuses on nutritional deficiencies in developing nations, global solutions for excesses and deficiencies need to be addressed. In an effort to achieve nutrition security, lifestyles, policies, and systems (eg, food, water, health, energy, education/knowledge, and economic) contributing to sustainable resource use, environmental management, health promotion, economic stability, and positive social environments are required. Food and nutrition practitioners can get involved in promoting and implementing effective and sustainable policies, systems, programs, and practices that support individual, community, and national efforts.

Anaemia, its correlation with overweight and growth patterns in children aged 5-10 years living in American Samoa.

OBJECTIVES: To determine the prevalence of anaemia, identify the predictors of anaemia, compare the prevalence of anaemia among children living in American Samoa to those found in children living in the USA, and compare the growth patterns obtained from this study to Centers for Disease Control and Prevention (CDC) data and data obtained earlier in American Samoan children. DESIGN: Cross-sectional. SETTING: American Samoa, a Pacific Island. SUBJECTS: In all, 208 children aged 5-10 years. RESULTS: Anaemia (Hb < 11.5 g/dl) prevalence was 17.3 %. There was a significant difference in mean Hb levels in children within American Samoa as compared to National Health and Nutrition Examination Survey III data (P < 0.05). In children with BMI Z-score (BMIZ) (P < 0.05) and weight-for-age Z-score (WAZ) (P < 0.05) >2.0, females had a significantly higher prevalence of anaemia than males. Females with a WAZ > 2.0 had a significantly higher prevalence of anaemia than females with a WAZ < or = 2.0 (P < 0.03). Risk factors for anaemia were mother having less than a high school education (P = 0.02), no car (P < 0.01) and no phone (P = 0.02). The BMIZ (P < 0.000), height-for-age Z-score (P < 0.000) and WAZ (P < 0.000) were significantly different from the distribution of CDC reference data and that found in children previously assessed in American Samoa. CONCLUSIONS: Anaemia is high among children aged 5-10 years living in American Samoa. Growth pattern Z-scores reveal that American Samoan children are, on average, taller, heavier and more overweight. Further examination into the causes of anaemia and overweight is warranted.

Mobile computing in the humanitarian assistance setting: an introduction and some first steps.

We developed a Palm operating system-based handheld computer system for admin istering nutrition questionnaires and used it to gather nutritional information among the Burmese refugees in the Mae La refugee camp on the Thai-Burma border Our experience demonstrated that such technology can be easily adapted for such an austere setting and used to great advantage. Further, the technology showed tremendous potential to reduce both time required and errors commonly encountered when field staff collect information in the humanitarian setting. We also identified several areas needing further development.

Iron deficiency is unacceptably high in refugee children from Burma.

Iron-deficiency anemia (IDA) in refugees is reported to be among the major medical problems worldwide. Because food rations are typically inadequate in iron, long-term reliance is a key predictor of anemia among displaced people. Comprehensive nutritional assessments of refugee children from Burma have not previously been completed. Refugee children aged 6-59 mo were studied to determine 1) the prevalences of anemia, iron deficiency (ID) and IDA and 2) the factors associated with anemia and ID. Cluster sampling in three camps and convenience sampling in two additional camps were used. Hemoglobin (Hb) levels were measured and micro mol zinc protoporphyrin/mol heme were determined in 975 children. Logistic regression analyses (95% CI) determined predictors of anemia and ID. The prevalences of IDA, anemia and ID in these refugee children were 64.9, 72.0 and 85.4%, respectively. Predictors of anemia included young age (P < 0.001), food ration lasting <1 mo (P = 0.001), daily consumption of dietary iron inhibitors (P < 0.05), weight-for-height Z-score of <-2 (P < 0.05), male gender (P < 0.05) and uneducated father (P < 0.001). Predictors of ID were young age (P < 0.001) and recently reported illness (P < 0.05). Laboratory tests confirmed that anemia and ID are major health problems among these refugee children and that ID is the leading cause of anemia. A comprehensive nutrition and public health-focused approach to combating anemia and ID is essential. Following the presentation of results to policy makers, the improvement of the micronutrient content of rations has been initiated.